Chronic hepatitis B (CHB) is caused by the Hepatitis B virus (HBV) and affects millions of people worldwide. Developing an effective CHB therapy requires using in vivo screening methods, such as mouse models reflecting CHB based on hydrodynamic delivery of plasmid vectors containing a replication-competent HBV genome. However, long-term expression of HBV proteins is accompanied by production of progeny virions, thereby requiring a Biosafety Level (BSL) 3 animal facility. In the present study, we introduced a point mutation in the START codon of the HBV polymerase to develop a mouse model reflecting chronic hepatitis B infection without formation of viral progeny. We induced the mouse model by hydrodynamic injection of adeno-associated virus plasmid vector (pAAV) and minicircle plasmid (pMC) constructs into C57Bl/6 and C3H/HeN mouse strains, monitoring HBV antigens and antibodies in blood by enzyme-linked immunosorbent assay and analyzing liver expression of HBV core antigen by immunohistology. Persisting expression of viral antigens over 140 days (study endpoint) was observed only in the C3H/HeN mouse strain when using pAAV/1.2HBV-A and pMC/1.0HBV-D with pre-C and pre-S recombination sites. In addition, pAAV/1.2HBV-A in C3H/HeN sustained HBV core antigen positivity up to the study endpoint in C3H/HeN mice. Moreover, introducing the point mutation in the START codon of polymerase effectively prevented the formation of viral progeny. Our study establishes an accessible and affordable experimental paradigm for developing a robust mouse model reflecting CHB suitable for preclinical testing of anti-HBV therapeutics in a BSL2 animal facility.

• MeSH
• chronická hepatitida B * genetika   MeSH
• kodon iniciační MeSH
• modely nemocí na zvířatech MeSH
• mutace MeSH
• myši inbrední C3H MeSH
• myši MeSH
• virus hepatitidy B genetika   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Chronic hepatitis B (CHB) remains a major public health problem worldwide, with limited treatment options, but inducing an antiviral response by innate immunity activation may provide a therapeutic alternative. We assessed the cytokine-mediated anti-hepatitis B virus (HBV) potential for stimulating the cyclic GMP-AMP synthase-stimulator of interferon genes (STING) pathway using STING agonists in primary human hepatocytes (PHH) and nonparenchymal liver cells (NPCs). The natural STING agonist, 2',3'-cyclic GMP-AMP, the synthetic analogue 3',3'-c-di(2'F,2'dAMP), and its bis(pivaloyloxymethyl) prodrug had strong indirect cytokine-mediated anti-HBV effects in PHH regardless of HBV genotype. Furthermore, STING agonists induced anti-HBV cytokine secretion in vitro, in both human and mouse NPCs, and triggered hepatic T cell activation. Cytokine secretion and lymphocyte activation were equally stimulated in NPCs isolated from control and HBV-persistent mice. Therefore, STING agonists modulate immune activation regardless of HBV persistence, paving the way toward a CHB therapy.

• MeSH
• cytokiny metabolismus   MeSH
• hepatitida B * farmakoterapie   MeSH
• hepatocyty MeSH
• herpesvirus B * MeSH
• interferony metabolismus   MeSH
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Cyclic dinucleotides (CDNs) trigger the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway, which plays a key role in cytosolic DNA sensing and thus in immunomodulation against infections, cell damage and cancer. However, cancer immunotherapy trials with CDNs have shown immune activation, but not complete tumor regression. Nevertheless, we designed a novel class of CDNs containing vinylphosphonate based on a STING-affinity screening assay. In vitro, acyloxymethyl phosphate/phosphonate prodrugs of these vinylphosphonate CDNs were up to 1000-fold more potent than the clinical candidate ADU-S100. In vivo, the lead prodrug induced tumor-specific T cell priming and facilitated tumor regression in the 4T1 syngeneic mouse model of breast cancer. Moreover, we solved the crystal structure of this ligand bound to the STING protein. Therefore, our findings not only validate the therapeutic potential of vinylphosphonate CDNs but also open up opportunities for drug development in cancer immunotherapy bridging innate and adaptive immunity.

• MeSH
• DNA MeSH
• imunoterapie MeSH
• myši MeSH
• nádory * farmakoterapie   MeSH
• nukleotidy cyklické * farmakologie   metabolismus   MeSH
• přirozená imunita MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Cyclic dinucleotides (CDNs) are second messengers that activate stimulator of interferon genes (STING). The cGAS-STING pathway plays a promising role in cancer immunotherapy. Here, we describe the synthesis of CDNs containing 7-substituted 7-deazapurine moiety. We used mouse cyclic GMP-AMP synthase and bacterial dinucleotide synthases for the enzymatic synthesis of CDNs. Alternatively, 7-(het)aryl 7-deazapurine CDNs were prepared by Suzuki-Miyaura cross-couplings. New CDNs were tested in biochemical and cell-based assays for their affinity to human STING. Eight CDNs showed better activity than 2'3'-cGAMP, the natural ligand of STING. The effect on cytokine and chemokine induction was also evaluated. The best activities were observed for CDNs bearing large aromatic substituents that point above the CDN molecule. We solved four X-ray structures of complexes of new CDNs with human STING. We observed π-π stacking interactions between the aromatic substituents and Tyr240 that are involved in the stabilization of CDN-STING complexes.

• MeSH
• cytokiny MeSH
• interferony MeSH
• lidé MeSH
• ligandy MeSH
• membránové proteiny * metabolismus   MeSH
• myši MeSH
• nukleotidy cyklické * chemie   MeSH
• nukleotidyltransferasy MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• Publikační typ
• tisková chyba MeSH

The cGAS-STING (cyclic GMP-AMP synthase-stimulator of interferon genes) pathway plays a crucial role in inducing an antiviral and antitumor immune response. We studied the effects of synthetic STING agonists on several immune populations and related cytokine production. In comparison with the toll-like receptor 7 (TLR7) agonist, STING agonists induced secretion of a broader proinflammatory cytokine spectrum. Unlike the TLR7 agonist, the structurally diverse STING agonists partially depleted B and NK cells and completely depleted CD14+ monocytes via induction of apoptosis. The TANK-binding kinase 1 inhibitor efficiently prevented interferon alpha (IFNα) secretion and cell depletion, suggesting their possible dependence on the cGAS-STING pathway activation. Finally, IFNα, tumor necrosis factor alpha, interleukin 6, and interleukin 1 beta secretion and CD14+ monocyte apoptosis were primary responses to STING agonists, whereas IFNγ was secreted secondarily. These findings bring new insights into the cGAS-STING pathway immunomodulation that is of future therapeutic importance.

• MeSH
• apoptóza MeSH
• cytokiny metabolismus   MeSH
• membránové proteiny genetika   MeSH
• monocyty * metabolismus   MeSH
• signální transdukce * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The oligoadenylate synthetase-ribonuclease L pathway is a major player in the interferon-induced antiviral defense mechanism of cells. Upon sensing viral dsRNA, 5'-phosphorylated 2',5'-oligoadenylates are synthesized, and subsequently activate latent RNase L. To determine the influence of 5'-phosphate end on the activation of human RNase L, four sets of 5'-phosphonate modified oligoadenylates were prepared on solid-phase. The ability of these 5'-modified oligoadenylates bearing shortened, isosteric and prolonged phosphonate linkages to activate RNase L was explored. We found that isosteric linkages and linkages prolonged by one atom were in general well tolerated by the enzyme with the EC50 values comparable to that of the natural activator. In contrast, linkages shortened by one atom or prolonged by two atoms exhibited decrease in the activity.

• MeSH
• adeninnukleotidy chemická syntéza   chemie   farmakologie   MeSH
• endoribonukleasy metabolismus   MeSH
• konformace nukleové kyseliny MeSH
• lidé MeSH
• oligoribonukleotidy chemická syntéza   chemie   farmakologie   MeSH
• organofosfonáty chemická syntéza   chemie   farmakologie   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The 3'-5', 3'-5' cyclic dinucleotides (3'3'CDNs) are bacterial second messengers that can also bind to the stimulator of interferon genes (STING) adaptor protein in vertebrates and activate the host innate immunity. Here, we profiled the substrate specificity of four bacterial dinucleotide synthases from Vibrio cholerae (DncV), Bacillus thuringiensis (btDisA), Escherichia coli (dgcZ), and Thermotoga maritima (tDGC) using a library of 33 nucleoside-5'-triphosphate analogues and then employed these enzymes to synthesize 24 3'3'CDNs. The STING affinity of CDNs was evaluated in cell-based and biochemical assays, and their ability to induce cytokines was determined by employing human peripheral blood mononuclear cells. Interestingly, the prepared heterodimeric 3'3'CDNs bound to the STING much better than their homodimeric counterparts and showed similar or better potency than bacterial 3'3'CDNs. We also rationalized the experimental findings by in-depth STING-CDN structure-activity correlations by dissecting computed interaction free energies into a set of well-defined and intuitive terms. To this aim, we employed state-of-the-art methods of computational chemistry, such as quantum mechanics/molecular mechanics (QM/MM) calculations, and complemented the computed results with the {STING:3'3'c-di-ara-AMP} X-ray crystallographic structure. QM/MM identified three outliers (mostly homodimers) for which we have no clear explanation of their impaired binding with respect to their heterodimeric counterparts, whereas the R2 = 0.7 correlation between the computed ΔG'int_rel and experimental ΔTm's for the remaining ligands has been very encouraging.

• MeSH
• Bacillus thuringiensis enzymologie   ultrastruktura   MeSH
• cytokiny chemie   genetika   MeSH
• Escherichia coli enzymologie   ultrastruktura   MeSH
• krystalografie rentgenová MeSH
• kvantová teorie MeSH
• leukocyty mononukleární chemie   enzymologie   MeSH
• lidé MeSH
• membránové proteiny chemie   genetika   ultrastruktura   MeSH
• nukleotidy biosyntéza   chemie   genetika   MeSH
• přirozená imunita genetika   MeSH
• substrátová specifita MeSH
• Thermotoga maritima enzymologie   ultrastruktura   MeSH
• Vibrio cholerae enzymologie   ultrastruktura   MeSH
• vztahy mezi strukturou a aktivitou * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Cyclic dinucleotides (CDNs) are second messengers that bind to the stimulator of interferon genes (STING) and trigger the expression of type I interferons and proinflammatory cytokines. Here we evaluate the activity of 3',3'-c-di(2'F,2'dAMP) and its phosphorothioate analogues against five STING allelic forms in reporter-cell-based assays and rationalize our findings with X-ray crystallography and quantum mechanics/molecular mechanics calculations. We show that the presence of fluorine in the 2' position of 3',3'-c-di(2'F,2'dAMP) improves its activity not only against the wild type (WT) but also against REF and Q STING. Additionally, we describe the synthesis of the acyloxymethyl and isopropyloxycarbonyl phosphoester prodrugs of CDNs. Masking the negative charges of the CDNs results in an up to a 1000-fold improvement of the activities of the prodrugs relative to those of their parent CDNs. Finally, the uptake and intracellular cleavage of pivaloyloxymethyl prodrugs to the parent CDN is rapid, reaching a peak intracellular concentration within 2 h.

• MeSH
• estery chemie   farmakologie   terapeutické užití   MeSH
• fosfáty chemie   metabolismus   farmakologie   terapeutické užití   MeSH
• HEK293 buňky MeSH
• interferon gama metabolismus   MeSH
• krystalografie rentgenová MeSH
• leukocyty mononukleární cytologie   účinky léků   metabolismus   MeSH
• lidé MeSH
• magnetická rezonanční spektroskopie MeSH
• membránové proteiny agonisté   metabolismus   MeSH
• prekurzory léčiv chemická syntéza   chemie   metabolismus   farmakologie   MeSH
• teorie funkcionálu hustoty MeSH
• TNF-alfa metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

STING protein (stimulator of interferon genes) plays an important role in the innate immune system. A number of potent compounds regulating its activity have been reported, mostly derivatives of cyclic dinucleotides (CDNs), natural STING agonists. Here, we aim to provide complementary information to large-scale "ligand-profiling" studies by probing the importance of STING-CDN protein-ligand interactions on the protein side. We examined in detail six typical CDNs each in complex with 13 rationally devised mutations in STING: S162A, S162T, Y167F, G230A, R232K, R232H, A233L, A233I, R238K, T263A, T263S, R293Q, and G230A/R293Q. The mutations switch on and off various types of protein-ligand interactions: π-π stacking, hydrogen bonding, ionic pairing, and nonpolar contacts. We correlated experimental data obtained by differential scanning fluorimetry, X-ray crystallography, and isothermal titration calorimetry with theoretical calculations. This enabled us to provide a mechanistic interpretation of the differences in the binding of representative CDNs to STING. We observed that the G230A mutation increased the thermal stability of the protein-ligand complex, indicating an increased level of ligand binding, whereas R238K and Y167F led to a complete loss of stabilization (ligand binding). The effects of the other mutations depended on the type of ligand (CDN) and varied, to some extent. A very good correlation (R2 = 0.6) between the experimental binding affinities and interaction energies computed by quantum chemical methods enabled us to explain the effect of the studied mutations in detail and evaluate specific interactions quantitatively. Our work may inspire development of high-affinity ligands against the common STING haplotypes by targeting the key (sometimes non-intuitive) protein-ligand interactions.

• MeSH
• bodová mutace * MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• membránové proteiny chemie   genetika   metabolismus   MeSH
• molekulární struktura MeSH
• nukleotidy cyklické chemie   metabolismus   MeSH
• proteinové domény MeSH
• vazebná místa MeSH
• vodíková vazba MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH